This application addresses issues related to 3' splice site recognition in the nematode C. elegans. Splicing in C. elegans requires recognition of very small introns which lack polypyrimidine tract and branch-point consensus sequences. The 3' splice site consensus, (U)4CAG/R, so far found uniquely in C. elegans, is required for both cis- and trans-splicing. The genes, uaf-1 and uaf-2, encode large and small subunits of the essential splicing factor U2AF, which are responsible for recognition of this 3' splice site consensus. Our aims are to determine using in vitro binding assays, the nature of the interaction between the (U)4CAG/R consensus and U2AF, and which domains of both subunits of U2AF are required for viability, in vitro binding, and activity. U2AF activity will be studied in vitro by complementation of a U2AF-depleted mammalian extract. In order to identify splicing components that interact with U2AF, mutants that suppress the dominant negative phenotype of part of U2AF expressed in the absence of the remainder of the protein will be selected. The gen encoding U2AF65, uaf-1, is alternatively spliced to include an extra exon which contains an array of ten copies of the (U)CAG/R consensus. The alternative splicing of uaf-1, pre-mRNA may be a way to tightly regulate levels of E2AF to insure accurate intron recognition. This idea will be tested by comparing levels of functional mRNA and extra-exon- containing RNA when U2AF is over-expressed. Mutants that change the relative levels of the two uaf-1 RNAs will be selected to determine how this alternative splicing event is regulated. The presence of this exon results in nuclear retention of RNA containing it. It will be determined how binding of USAF to the (U)4CAG/R elements causes nuclear retention. In order to identify additional components of the machinery needed to prevent export of RNA containing these sequences, mutants that suppress the nuclear retention phenotype will be selected in C. elegans.